Methods and pharmaceuticals for treatment of viral infections of the eye

ABSTRACT

Viral infections of the eye, and particularly viral infections in the Herpesviridae and Adenoviridae families, can be treated by administration of a pharmaceutical made up of an enzymatically active ribonuclease and a vehicle. Advantageously, the enzymatically active ribonuclease is ranpirnase, the &#39;805 variant, rAmphinase 2, and Amphinase 2, and the vehicle is an aqueous solution.

BACKGROUND OF THE INVENTION

The invention relates to viral infections of the eye, and moreparticularly relates to methods and pharmaceuticals for treatment ofviral infections of the eve. In its most immediate sense, the inventionrelates to treatment of human eye infections caused by Herpesviridaeviruses (including but not limited to Human cytomegalovirus, herpeszoster virus, and varicella zoster virus) and Adenoviridae viruses.Viral diseases of the eye can have significant consequences. Type 1herpes simplex virus can cause conjunctivitis and keratitis, Humancytomegalovirus can cause retinitis, adenovirus types 8, 19, 29, and 37can cause epidemic keratoconjunctivitis, and adenovirus types 3, 4, and7 can cause pharyngoconjuctival fever. Herpes zoster virus (HZV), amember of the Herpesviridae family, can cause severe eye disease whenaffecting the trigeminal area. Herpes zoster ophthalmicus, a severe formof acute herpes zoster, results from the reactivation of varicellazoster virus (VZV) (another member of the Herpesviridae family) in thetrigeminal (fifth cranial) nerve. Any branch of the nerve may beaffected, though the frontal branch within the first division of thetrigeminal nerve is most commonly involved. This frontal branchinnervates nearly all of the ocular and periocular structures. Herpeszoster ophthalmicus at this particular location can lead to blindnessand requires a fast and effective therapeutic approach.

Human cytomegalovirus (CMV) is another member of the Herpesviridaefamily. At least 60% of the US population has been exposed to CMV, witha prevalence of more than 90% in high⁻risk groups (e.g., unborn babieswhose mothers become infected with CMV during pregnancy, people withHIV, and transplant recipients).

CMV retinitis is one of the most common opportunistic infections inpersons with AIDS or pharmacologically induced immunosuppression.Individuals with CMV retinitis typically exhibit a progressive decreasein visual acuity, which may progress to blindness. Long-term CMVtreatment is necessary to prevent retinitis relapse.

Immune reconstitution syndrome (IRIS) is reported in 16-63% ofHIV-infected patients with CMV retinitis following the initiation ofHAART (Highly Active Antiretroviral Treatment). CMV IRIS may manifest aspainless floaters, blurred vision, photopia, decreased visual acuity, orocular pain. Some Patients may develop macular edema leading to visionloss or proliferative vitreoretinopathy, spontaneous vitreal hemorrhage,and retinal detachment.

It is known to treat viral eye infections with acyclovir but suchtreatment is not entirely satisfactory. Topical acyclovir must beapplied frequently and causes irritation of the eye. Oral acyclovircauses significant adverse side effects. Other antiviral medicationssuch as gancyclovir, valacyclovir and valgancyclovir are used to treatviral eye infections, and such treatments are also not entirelysatisfactory. Gancyclovir is administered intravenously, and thereforecannot be used outside e.g. a hospital setting. Oral antiviralmedications such as valacyclovir and valgancyclovir have disadvantages;they are known to cause fever, rash, diarrhea, and hematologic effects(e.g., neutropenia, anemia, thrombocytopenia). In some cases neutropeniamay respond to lowering the dose or using drugs that stimulate theproduction of neutrophils by the bone marrow as granulocytecolony-stimulating factor [G-CSF], or granulocyte-macrophagecolony-stimulating factor [GM-CSF]. These toxic effects can be difficultto manage.

It would therefore be advantageous to provide a better method and abetter pharmaceutical for treating viral eye infections in humans.

Various enzymatically active ribonucleases, including ranpirnase andother proteins that are highly homologous to it, are known to haveantiviral activity, and to have activity against viruses in theHerpesviridae family (specifically including but not limited to Herpessimplex virus types 1 and 2 and Human cytomegalovirus) and also againsttype 2 adenovirus. However, proteins are known to be highly irritatingto the eye due to an intense inflammatory response mediated by T-cells.For this reason, although ranpirnase and other related proteins havebeen investigated for use against various viral infections, they havenot been investigated for use against viral infections of the eye.

Despite the expectation that proteinaceous ranpirnase would causeirritation in the eye, irritation of topically applied ranpirnase in theeye was studied in a rabbit model. In this experiment, ranpirnase wasdemonstrated to be non-irritating as determined using the GloballyHarmonized System of Classification Evaluation Criteria and the EuropeanEconomic Community Ocular Evaluation Criteria. This was a remarkableresult, because administration of a foreign protein to the eye canproduce corneal irritation. As a result, ranpirnase and other proteinsthat are highly homologous to it are expected to be useful in treatingviral infections of the human eye.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the followingexemplary and non-limiting drawings, in which:

FIG. 1 shows the scale used for scoring ocular lesions observed in arabbit that has undergone a Draize test;

FIG. 2 shows the results of a Draize test in which a ranpirnase solutionwas applied to the right eye of three rabbits;

FIG. 3 shows the results of the Draize test of FIG. 2 in which the lefteye of each of the rabbits was untreated; and

FIG. 4 shows the European Economic Community Ocular Evaluation Criteriaused to classify the ocular irritation caused by a test article in aDraize test.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Ranpirnase is a proteinaceous enzymatically active ribonuclease (theword “ribonuclease” is frequently abbreviated as “RNase”) that isdisclosed and claimed in U.S. Pat. No. 5,559,212. U.S. Pat. Nos.5,728,805, 6,239,257, 7,229,824 and U.S. Pay. No. 8,518,399 disclosethree other proteinaceous enzymatically active ribonucleases that arehighly homologous to ranpirnase:

a) the ribonuclease of SEQ ID NO:2 in U.S. Pat. No. 5,728,805, hereinreferred to as: the “'805 variant”;

b) the ribonuclease of SEQ ID NO:2 in U. S. Pat. No. 6,239,257, hereinreferred to as “Amphinase 2”, and;

c) the ribonuclease of SEQ ID NO:59 of U.S. Pat. No. 7,229,824, hereinreferred to as “rAmphinase 2”.

U.S. Pat. No. 8,518,399 discloses that ranpirnase, the '805 variant, andrAmphinase 2 have antiviral activity against Herpesviridae viruses,specifically including but not limited to Herpes simplex types 1 and andHuman cytomegalovirus. Based upon its similarities with these threeenzymatically active ribonucleases, it is believed that Amphinase 2 willhave these activities as well.

Commonly-owned copending patent application Ser. No. 14/736,170 filedJun. 10, 2015 and published as US 2015/0376584 A1 discloses thatranpirnase has antiviral activity against a number of viruses, includingtype 2 adenovirus. As stated therein, the viruses in the Adenoviridaefamily are very closely related and the demonstrated antiviral activityof ranpirnase against any one virus within the Adenoviridae family isstrong evidence that ranpirnase will have the same anti-replicationactivity against all viruses within the Adenoviridae family.Furthermore, as stated in that pending patent application, to a personof ordinary skill in this art, the similarities of homology and activityof these three other ribonucleases is strong evidence that these threeother ribonucleases will have the same activity as ranpirnase has.Hence, although in vitro experiments have not yet been repeated usingthe '805 variant, Amphinase 2, or rAmphinase 2, a person of ordinaryskill in this art would conclude that these three ribonucleases willlikewise be active against all the viruses in the Adenoviridae family.

Thus, ranpirnase, the '805 variant, Amphinase 2, and rAmphinase 2 haveeither been demonstrated to be active against type 1 Herpes simplexvirus and viruses in the Adenoviridae family or would be expected to beso active. For this reason, a person of ordinary skill in the art wouldexpect that all four of these proteinaceous enzymatically activeribonucleases will be useful against viral infections of the human eye.

All four of these ribonucleases are members of the ribonuclease Asuperfamily. Such ribonucleases are pyrimidine-specific endonucleasesfound in high quantity in the pancreas of certain mammals and of somereptiles. They are involved in endonucleolytic cleavage of3′-phosphomononucleotides and 3′-phosphooligonucleotides ending in C-Por U-P with 2′,3′-cyclic phosphate intermediates. Other members of thissuperfamily include bovine seminal vesicle and brain ribonucleases,kidney non-secretory ribonucleases, liver-type ribonucleases,angiogenin; eosinophil cationic protein, pancreatic ribonucleases fromdifferent species including human, and bovine pancreatic ribonucleases.

Ranpirnase, which is disclosed in U.S. Pat. No. 5,559,212, and waspreviously known by the ONCONASE trademark, is a ribonuclease isolatedfrom oocytes of the leopard frog Rana pipiens. The amino acid sequenceof ranpirnase is provided in SEQ ID NO: 1. Ranpirnase has been testedand found to be cytotoxic to cancer cells because of its enzymaticactivity against RNA.

A variant of ranpirnase (hereinafter, the “'805 variant”) is disclosedin U.S. Pat. No. 5,728,805. The '805 variant is also a ribonuclease, andhas likewise been found to be cytotoxic to certain cancer cells. The'805 variant is a close variant of ranpirnase; its amino acid sequenceis identical to that of ranpirnase except that it has valine instead ofisoleucine at position 11, asparagine instead of aspartic acid atposition 20, and arginine instead of serine at position 103 of theranpirnase amino acid sequence. The '805 variant has been referred to as“Valli, Asn20, Arg103-Ranpirnase”. The amino acid sequence of the '805variant is provided in SEQ ID NO:2.

Amphinase 2 is also a ribonuclease. It is the protein identified as2325p4 in U.S. Pat. No. 6,239,257 and it too has been found to becytotoxic to cancer cells. The amino acid sequence of Amphinase 2 isprovided in SEQ ID NO: 3.

Recombinant Amphinase 2 (“rAmphinase 2”) is similar to Amphinase 2, buthas a Net residue at position −1 and lacks glycan moieties that arelocated in Amphinase 2 at positions 27 and 91. rAmphinase 2 is describedin U.S. Pat. No. 7,229,824. The amino acid sequence of rAmphinase 2 isprovided in SEQ ID NO: 4.

The term “functional equivalent” is intended to mean any protein thatdiffers from any naturally occurring ribonuclease by the deletion,addition or substitution of one or more amino acids, but that retainsribonuclease activity. For example, the '805 variant is a functionalequivalent of ranpirnase, because it comprises three amino acidsubstitutions compared to the ranpirnase amino acid sequence, but stillhas RNase activity.

The disclosures of U.S. Pat. Nos. 5,559,212, 5,728,805, 6,239,257,7,229,824, 8,518,399, 8,663,964 and published patent application US2015/0376584 A1 are all incorporated by reference herein in theirentireties for all purposes.

As discussed above, it has been found that a ribonuclease of the RNase Asuperfamily, in particular ranpirnase, can be used in the treatment of aviral infection of the eye. A “viral infection of the eye” is a diseasewhich shows symptoms predominantly in the eye of a subject and which iscaused by viruses rather than bacteria. Viral diseases of the eyeinclude, but are not limited to, conjunctivitis and keratitis,retinitis, keratoconjunctivitis, chorioretinitis, pharyngoconjuctivalfever and CMV retinitis.

Viral conjunctivitis, also known as pink eye, is characterized byinflammation of the outermost layer of the white part of the eye and theinner surface of the eyelid. Viral conjunctivitis is typically caused byan adenovirus. Other viruses that can be responsible for conjunctivalinfection include herpes simplex virus (HSV), varicella-zoster virus(VZV), enterovirus 70, Coxsackie virus A24, molluscum contagiosum andhuman immunodeficiency virus (HIV).

Viral keratitis is an inflammation of the cornea which is predominantlycaused by herpes simplex virus.

Chorioretinitis is an inflammation of the choroid (thin pigmentedvascular coat of the eye) and retina of the eye. It is a form ofposterior uveitis. Chorioretinitis can be caused by infection withcytomegalovirus (CMV), Varicella-Zoster (HZV), dengue fever, West Nilevirus or lymphocytic choriomeningitis virus (LCMV).

In particular, viral infections of the eye can be caused by a virus fromthe Herpesviridae family of viruses and by an adenovirus selected fromthe group consisting of 3, 4, 7, 8, 19, 29, and 37. Herpesviridae areviruses having a double-stranded, linear DNA genome which are classifiedin Baltimore class I. Viruses from the Herpesviridae family causingviral infections of the eye include, but are not limited to, type IHerpes simplex virus, human cytomegalovirus and Herpes zoster virus, inparticular Herpes zoster ophthalmicus.

Adenoviridae are double-stranded DNA viruses which are classified inBaltimore class I. Adenovirus types 8, 19, 29, and 37 can cause epidemickeratoconjunctivitis, and adenovirus types 3, 4, and 7 can causepharyngoconjuctival fever types 8, 19, 29, and 37 can cause epidemickeratoconjunctivitis, and Adenovirus types 3, 4, and 7 can causepharyngoconjuctival fever. The terms “treating” and “treatment,” as usedherein, refer to administering to a subject having a viral infection ofthe eye a therapeutically effective dose of a ribonuclease such asranpirnase, a ranpirnase variant such as the '805 variant, Amphinase 2,or rAmphinase 2. As used herein, the term “treating” covers anytreatment of a viral infection of the eye which results in a desiredpharmacologic and/or physiologic effect, including arresting diseasedevelopment, causing regression of the disease, limiting spread of thevirus from one cell to another within an individual, limitingreplication of a virus in an individual, limiting entry of a virus intothe cell of an individual and reducing the number of viruses in anindividual or a tissue of this individual.

The term “therapeutically effective dose” refers to an amount of apharmaceutical that results in an improvement or remediation of thesymptoms of a disease or condition to be treated. A therapeuticallyeffective dose of a ribonuclease such as ranpirnase, '805 variant,Amphinase 2, or rAmphinase 2, delays or minimizes the onset of, orhastens or increases recovery of a subject from, a viral infection ofthe eye in a subject. The RNase may reduce the viral titer in the eye ofthe infected subject, or may prevent the viral titer in the eye of theinfected subject from increasing. A therapeutically effective dose of aribonuclease may provide a therapeutic benefit in the treatment ormanagement of a viral infection of the eye by reducing the spread of thevirus from one cell to another and may also prevent disease and/orreduce the severity of symptoms.

A therapeutically effective dose can be determined by the skilled personas a matter of routine experimentation. The therapeutically effectivedosage of the pharmaceutical composition can be determined readily bythe skilled artisan, for example, from animal studies. In addition,human clinical studies can be performed to determine the preferredeffective dose for humans by a skilled artisan. Such clinical studiesare routine and well known in the art. The precise dose to be employedwill also depend on the route of administration. Effective doses may beextrapolated from dose-response curves derived from in vitro or animaltest systems.

The RNase may be administered to a subject in need thereof in a singledose or in multiple doses. The RNase may be administered to the subjectuntil symptoms resolve and/or until the subject is no longer at risk ofa virus infection. The schedule on which the administration of the RNase(advantageously, ranpirnase) commences, and on which a single dose ormultiple doses of the RNase (advantageously, ranpirnase) is or areadministered to the subject, and the duration of dosing, can bedetermined by a person of ordinary skill. These factors may depend onfactors such as the severity of symptoms, patient response, etc.

The administration of a therapeutically effective dose of the RNase(advantageously, ranpirnase) may reduce the virus titer in the eyecompared to a control that is infected with the virus but not treatedwith the RNase.

The administration of a therapeutically effective dose of the RNase(advantageously, ranpirnase) may lead to a reduction of the virus titerbelow the detection level. Determination of virus titers is for examplediscussed in Reischl (1996) Front Biosci. 1:e 72-7, Application ofmolecular biology-based methods to the diagnosis of infectious diseases.

The ribonuclease (advantageously, ranpirnase) is preferably administeredtopically to the eye, i.e. is administered directly to the eye and notelsewhere on the body. The topical administration may be by e.g. eyedrops, a suspension, an emulsion, an ointment, a solution, a gel,liposomes, nanoparticles, microemulsions, nanoemulsions,nanosuspensions, niosomes, dendrimers and hydrogels. Alternatively, theribonuclease (advantageously, ranpirnase) may be administered by theintravitreal, intracameral, subconjunctival, subtenon, retrobulbar orposterior juxtascleral route.

The ribonuclease will be a component of a pharmaceutical that includes avehicle. The term “vehicle” includes any and all solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents and the like. The use of such media andagents for pharmaceutically active substances is well known in the art.These agents are generally safe, non-toxic and neither biologically norotherwise undesirable.

The vehicle may contain ingredients such as an excipient, a surfactant,an ingredient to adjust the pH of the composition and to buffer within acertain pH range, and a tonicity agent to adjust the tonicity of thecomposition. Such ingredients are known, and persons of ordinary skillin the art can select such of them as will produce a formulation havingappropriate characteristics when combined with the RNase(advantageously, ranpirnase).

It is possible to determine whether a substance (here, a ribonuclease)is non-irritating to the eye in a Draize test using the GloballyHarmonized System of Classification Evaluation Criteria and using theEuropean Economic Community Ocular Evaluation Criteria. In the Draizetest the RNase is placed in the conjunctival sac of a rabbit's eye andthe eye is examined at 1, 24, 48 and 72 hours after instillation ofranpirnase. The criteria evaluated are corneal opacity, iris lesion,conjunctival redness and conjunctival edema. If the score for each ofthese criteria is zero, the tested substance is considerednon-irritating to the eye. FIGS. 1 and 4 show, respectively, the scaleused for scoring ocular lesions observed in a rabbit that has undergonea Draize test and the European Economic Community Ocular EvaluationCriteria used to classify the ocular irritation caused by a test articlein a Draize test.

As stated above, before the present invention, no person of ordinaryskill in the art would administer to the eye ranpirnase or any of theother three above-identified proteinaceous enzymatically activeribonucleases. However, such administration of ranpirnase has beenmodeled using the Draize test and the results of this experimentdemonstrate that ranpirnase is non-irritating as defined by two acceptedstandards.

EXAMPLE

A 0.1% mL solution made up of 0.1% ranpirnase in a proprietary aqueoussolution used as a vehicle was used as a test article. Three rabbitswere used; each was a male New Zealand White rabbit that wasapproximately 16 weeks old at the time of the experiment and thatweighed 3.3 to 3.4 kg.

After administration of two drops of Tetracaine pre-anesthetic to thecorneal surface of both eyes of each rabbit, the test article was placedin the conjunctival sac of the right eye of each rabbit by gentlypulling the lower lid away from the eyeball; the lids were gently heldtogether for approximately one second to limit the loss of the testmaterial. The left eye of each rabbit remained untreated and served asthe control. The eyes of the animals were examined at 1 (±15 minutes),24, 48, and 72 hours (±1 hour) after installation of the test article.The grades of ocular reaction according to Draize (FIG. 1) were manuallyrecorded at each examination (FIGS. 2 and 3). As can be seen in FIG. 2,there was an ocular reaction in each animal one hour post-instillationof the test article but in every instance that reaction was completelyresolved by 24 hours and thereafter.

To determine the degree of irritation caused by the test article usingthe European Economic Community Ocular Evaluation Criteria (FIG. 4), thetotal ocular irritation scores for the examinations at 24, 48, and 72hours were individually added for corneal opacity, iris lesion,conjunctival redness, and conjunctival edema and the mean scores forthese scoring parameters were compared to the European EconomicCommunity Ocular Evaluation Criteria. Because all these scores (andtherefore the calculated mean scores) were zero, the test subject wasconsidered to be non-irritating as defined by the European EconomicCommunity Ocular Evaluation Criteria.

To determine the degree of irritation caused by the test article usingthe Globally Harmonized System of Classification Evaluation Criteria,the 24-, 48-, and 72-hour scores were added separately for each animaland each total divided by 3 (three time points) to yield the individualmean scores for each animal. Because all these scores (and therefore thecalculated quotients) were zero, the test subject was considered to benon-irritating as defined by the Globally Harmonized System ofClassification Evaluation Criteria.

Hence, these test data demonstrate a new and unexpected result:ranpirnase delivered to the eye in an aqueous solution is non-irritatingas defined by the Globally Harmonized System of ClassificationEvaluation Criteria and by the European Economic Community OcularEvaluation Criteria, even though ranpirnase is a protein (which would beexpected to be irritating to the eye).

Although this experiment was carried out using a solution of ranpirnasein a proprietary aqueous vehicle, a person of ordinary skill in the artwould consider it likely that solutions of the three above-identifiedproteinaceous enzymatically active ribonucleases would behave in thesame way because of their similarities to ranpirnase in respect ofactivity and homology.

Although at least one preferred embodiment of the invention has beendescribed above, this description is not limiting and is only exemplary.The scope of the invention is defined only by the claims, which follow:

1. A method of treating a viral infection of the eye, comprising thestep of administering to the eye a therapeutically effective dose of aribonuclease that is a member of the ribonuclease A superfamily.
 2. Themethod of claim 1, wherein the eye is a human eye and the ribonucleaseis selected from a group consisting of a. ranpirnase; b. the '805variant; c. Amphinase 2; and d. rAmphinase
 2. 3. The method of claim 1,wherein the viral infection is: a. in the Herpesviridae family ofviruses; or b. in the Adenoviridae family of viruses.
 4. The method ofclaim 1, wherein the virus is a. Human cytomegalovirus; or b. Herpessimplex virus.
 5. The method of claim 1, wherein the ribonuclease isnon-irritating to the eye as determined using a. the Globally HarmonizedSystem of Classification Evaluation Criteria and b. the EuropeanEconomic Community Ocular Evaluation Criteria.
 6. The method of claim 1,wherein the ^(eye) is a human eye and the ribonuclease is functionallyequivalent to a ribonuclease selected from a group consisting of: b. the'805 variant; c. Amphinase 2; and d. rAmphinase
 2. 7. A pharmaceuticalfor treating a viral infection of the human eye, comprising: a. aribonuclease that is a member of the ribonuclease A superfamily and thatis non-irritating to the eye as determined using i. the GloballyHarmonized System of Classification Evaluation Criteria and ii. theEuropean Economic Community Ocular Evaluation Criteria; and b. avehicle.
 8. The pharmaceutical of claim 7, wherein the ribonuclease isselected from a group consisting of: a. ranpirnase; b. the '805 variant;c. Amphinase 2; and d. rAmphinase
 2. 9. The pharmaceutical of claim 7,wherein the infection is: a. in the Herpesviridae family of viruses; orb. in the Adenoviridae family of viruses.
 10. The pharmaceutical ofclaim 7, wherein the virus is a. Human cytomegalovirus; or b. Herpessimplex virus.
 11. The pharmaceutical of claim 7, wherein the vehicle isan aqueous solution.
 12. The pharmaceutical of claim 7, wherein theribonuclease is the functional equivalent of a ribonuclease selectedfrom a group consisting of a. ranpirnase; b. the '805 variant; c.Amphinase 2; and d. rAmphinase 2.